Proteome profiler human cell stress array kit

In the primate tissue snap frozen retinal samples were either used to assess the levels of cytokine using the Proteome Profiler Human Cytokine Array Panel A (R & D Systems, Minneapolis, MN, USA) or the levels of cell stress-related proteins using the Human Cell Stress Proteome Profiler Array kit (R&D Systems, Minneapolis, MN, USA). While these arrays are for human, at both gene and protein levels there is a 93–99% homology between human and Macaque cytokines^{73 (link)}. Retinae were pooled from defined retinal regions in different primates and across whole retinae in mice. The retinae were homogenized in RIPA buffer (Millipore, UK) containing protease inhibitors (Sigma, UK). Lysates were centrifuged at 1000 × g for 10 minutes at 4 °C and the supernatant was used for the arrays and were conducted according to the manufacturer’s instructions and offer a parallel determination of 36 cytokines for the cytokine array and 26 for the human cell stress related proteins. Protein concentration was calculated using the Protein Assay (Thermo Scientific). Protein Array Analyzer for Image J was used to quantify and determine spot density. In the mouse tissues snap frozen retinae were used to assess the levels of cytokine using the Proteome Profiler Mouse Cytokine Array Panel A (R & D Systems, Minneapolis, MN, USA).

Protein arrays were performed using a Proteome Profiler Human Cell Stress Array Kit (R&D Systems). Briefly, BeWo cell lysates were incubated with the arrays at 4°C overnight and proteins were detected according to the manufacturer's instructions. Dots were detected using X-ray film and densitometric analysis was carried out using Image J software.